Project Summary The long-term goal of this project is to determine how mutations in the human LMNA gene encoding the nuclear envelope (NE) proteins lamin A and C cause severe cardiac and skeletal muscle diseases. This research is important because mutations in the LMNA gene are responsible for approximately 10% of all genetically inherited cases of dilated cardiomyopathy, and LMNA-associated cardiomyopathies have a particularly poor prognosis. Individuals with LMNA-associated muscular dystrophy suffer from debilitating progressive muscle wasting and die from dilated cardiomyopathy. To date, no effective treatments are available for the cardiac and skeletal muscle defects caused by LMNA mutations, and it remains unclear how LMNA mutations result in muscle- specific defects. The incomplete understanding of the disease pathogenesis presents a major hurdle in the development of effective treatments. Building on recently published and extensive preliminary data, this project proposes a novel hypothesis for the pathogenesis of laminopathies affecting cardiac and skeletal muscle: LMNA mutations associated with striated muscle disease reduce nuclear mechanical stability. In skeletal and cardiac tissues, the fragile nuclei are then prone to mechanically induced damage to the NE (?NE rupture?) due to cytoskeletal forces acting on myonuclei during muscle cell contraction and maturation. The NE rupture results in DNA damage and activation of DNA damage response pathways, which lead to cell death, senescence, and metabolic impairment responsible for the progressive cardiac and skeletal muscle defects. Additional mechanisms, such as disturbed transcriptional regulation and increased production of reactive oxygen species, may further contribute to the increased DNA damage and activation of DNA damage response pathways in the LMNA mutant muscle cells. The proposed work takes advantage of several in vitro and in vivo models and assays that were custom-developed in the PI?s laboratory to measure nuclear mechanics and mechanically induced damage in living cells and fixed tissues. The proposed project aims to: (1) determine the molecular and biophysical mechanisms that cause the increased NE rupture, DNA damage, and DNA damage response activation in skeletal and cardiac muscle cells in laminopathies; (2) identify the molecular pathways leading from NE rupture, DNA damage, and DNA damage response activation to striated muscle cell death and dysfunction; and (3) evaluate if inhibiting hyperactive DNA damage response pathways or reducing physical stress on the nucleus improves cardiac and skeletal muscle health in mouse models of LMNA-associated disease. The proposed studies will establish the functional role of nuclear damage observed in laminopathies and provide new insights into the pathogenesis of laminopathies affecting cardiac and skeletal muscle, with the potential to discover novel therapeutic targets and strategies that could significantly enhance the treatment options available for affected individuals.